Exogenous surfactant reduces inflammation and redox imbalance in rats under prone or supine mechanical ventilation.

Mechanical ventilation (MV) is a lifesaving therapy for patients with acute or chronic respiratory failure. Despite, it can also cause lung injury by inducing or worsening inflammatory responses and oxidative stress. Several clinical approaches have protective effects on the lungs, including the prone position and exogenous surfactant; however, few studies have evaluated the association between the two strategies, especially in individuals without previous lung injury. We tested the hypothesis that the effects of the homogenization in lung aeration caused by the prone position in association with the anti-inflammatory properties of exogenous surfactant pre-treatment could have a cumulative protective effect against ventilator-induced lung injury. Therefore, Wistar rats were divided into four experimental groups: Mechanical Ventilation in Supine Position (MVSP), Mechanical Ventilation in Prone position (MVPP), Mechanical Ventilation in Supine Position + surfactant (MVSPS), and Mechanical Ventilation in Prone Position + Surfactant (MVPPS). The intranasal instillation of a porcine surfactant (Curosurf®) was performed in the animals of MVSPS and MVPPS 1 h before the MV, all the rats were subjected to MV for 1 h. The prone position in association with surfactant decreased mRNA expression levels of pro-inflammatory cytokines in ventilated animals compared to the supine position; in addition, the NfκB was lower in MVPP, MVSPS and MVPPS when compared to MVSP. However, it had no effects on oxidative stress caused by MV. Pre-treatment with exogenous surfactant was more efficient in promoting lung protection than the prone position, as it also reduced oxidative damage in the lung parenchyma. Nevertheless, the surfactant did not cause additional improvements in most parameters that were also improved by the prone position. Our results indicate that the pre-treatment with exogenous surfactant, regardless of the position adopted in mechanical ventilation, preserves the original lung histoarchitecture, reduces redox imbalance, and reduces acute inflammatory responses caused by mechanical ventilation in healthy adult Wistar rats.

Immunoproteomics approach for the discovery of antigens applied to the diagnosis of canine visceral leishmaniasis.

In the present study, an immunoproteomic approach using Leishmania infantum parasites isolated from naturally infected dogs from an endemic region of the disease, was carried out to identify new antigens to be used in the diagnosis of canine visceral leishmaniasis (CVL). Protein extracts, obtained from parasites isolated from asymptomatic (CanLA) and symptomatic (CanLS) dogs, were used to perform the two-dimensional gels. Western Blotting assays were carried out by employing a pool of sera from dogs with visceral leishmaniasis (CanLA or CanLS), healthy dogs from an endemic area, or dogs with similar diseases associated with cross-reactions (babesiosis and ehrlichiosis). With these results, it was possible to exclude the spots that showed a cross-reactivity of the sera from groups of healthy dogs, and those with babesiosis or ehrlichiosis. Taken together, 20 proteins were identified, 15 of which have already been described in the literature and 5 of which are hypothetical. An immunogenomic screen strategy was applied to identify conserved linear B-cell epitopes in the identified hypothetical proteins. Two peptides were synthesized and tested in ELISA experiments as a proof of concept for the validation of our immunoproteomics findings. The results demonstrated that the antigens presented sensitivity and specificity values ranging from 81.93% to 97.59% and 78.14 to 85.12%, respectively. As a comparative antigen, a preparation of a Leishmania extract showed sensitivity and specificity values of 75.90% and 74.88%, respectively. The present study was able to identify proteins capable of being used for the serodiagnosis of canine visceral leishmaniasis.

The Use of an Adjuvant System Improves Innate and Adaptive Immune Response When Associated with a Leishmania (Viannia) braziliensis Antigen in a Vaccine Candidate against L. (Leishmania) infantum Infection.

BACKGROUND: The adjuvants' optimal dose and the administration route can directly influence the epitope recognition patterns and profiles of innate response. We aimed to establish the effect and the optimal dose of adjuvant systems for proposing a vaccine candidate to be employed with Leishmania (Viannia) braziliensis. METHODS: We evaluated the adjuvants saponin (SAP), monophosphoryl lipid A (MPL) and resiquimod (R-848) isolated and combined as adjuvant systems in a lower dose corresponding to 25%, 33%, and 50% of each adjuvant total dose. Male outbred BALB/c mice were divided into 13 groups, SAP, MPL, and R-848 isolated, and the adjuvant systems SAP plus MPL (SM), SAP plus R-848 (SR), and MPL plus R-848 (MR). RESULTS: SM50 increased levels of all chemokines analyzed and TNF production, while it presented an increased inflammatory cell infiltrate in the skin with macrophage recruitment. Thus, we proposed a vaccine candidate employing L. (V.) braziliensis antigen associated with the SM adjuvant system against experimental L. (Leishmania) infantum challenge. We observed a significant increase in the frequency of cells expressing the central and effector memory CD4+ T cells phenotype in immunized mice with the LBSM50. In the liver, there was a decreased parasite load when mice received LBSM50. CONCLUSIONS: When combined with L. (V.) braziliensis antigen, SM50 increases TNF and IFN-γ, which generates central and effector memory CD4+ T cells. Therefore, using an adjuvant system can promote an effective innate immune response with the potential to compose future vaccines.

CD4+ T-lymphocytes from asymptomatic dogs infected with Leishmania infantum are able to activate macrophages for higher leishmanicidal ability in an in vitro co-culture experiment.

Dogs are the most common domestic reservoir of Leishmania infantum, making canine visceral leishmaniasis (CVL) a serious public health issue. Identifying new methodologies that can mimic lymphoid and myeloid competence in naturally infected dogs could lower costs and save time in preliminary screenings of potential immunotherapeutic agents and vaccines against CVL. For that, we established a cell-to-cell communication approach between lymphocytes and myeloid cells from healthy, asymptomatic (infected, without apparent clinical signs) and symptomatic (infected with apparent clinical signs) dogs. Peripheral blood mononuclear cells (PBMC) from these dogs were used as source of CD4+, CD8+ T lymphocytes and macrophages, that were posteriorly infected with L. infantum GFP+ promastigotes (green fluorescent protein). Macrophages co-cultured with purified lymphocytes were tested for the ability to control cellular parasitism, and their microbicidal function by producing nitric oxide (NO) and reactive oxygen species (ROS). The kind of T cell response within the co-culture was also evaluated, by assessing their ability to produce interferon-gamma (IFN-γ) and interleukin 4 (IL-4). The data suggests that T lymphocytes from symptomatic dogs are more prone to produce IL-4 than the ones from asymptomatic dogs. Macrophages from asymptomatic dogs also demonstrated a higher microbicidal potential, with increased levels of NO and ROS production, compared to symptomatic dogs, mainly in highly parasitized cells. Together, our results identify the ratio of IL-4/IFN-γ produced by CD4+ and CD8+ T cells, as well as, the ratio between parasite GFP signal/NO and ROS signal in macrophages as potential immunological biomarkers of failure and success of the screened agents. Our findings also propose a reliable methodology that can be used to follow the immune response in trials of potential drugs or vaccines targeting CVL.

LBMPL Vaccine Therapy Induces Progressive Organization of the Spleen Microarchitecture, Improved Th1 Adaptative Immune Response and Control of Parasitism in Leishmania infantum Naturally Infected Dogs.

The spleen plays a central role in human and canine visceral leishmaniasis, where the activation of the immune response occurs in one of the tissues where Leishmania infantum reproduces. Therefore, this organ is both a target to understand the mechanisms involved in the parasite control and a parameter for assessing the therapeutic response. In this sense, this study aimed to evaluate the main histological, immunological and parasitological aspects in the spleen of symptomatic dogs naturally infected by L. infantum treated with the therapeutic vaccine LBMPL. For this, dogs were divided into four groups: dogs uninfected and untreated (NI group); L. infantum-infected dogs that were not treated (INT group); L. infantum-infected dogs that received treatment only with monophosphoryl lipid A adjuvant (MPL group); and L. infantum-infected dogs that received treatment with the vaccine composed by L. braziliensis promastigote proteins associated with MPL adjuvant (LBMPL group). Ninety days after the therapeutics protocol, the dogs were euthanized and the spleen was collected for the proposed evaluations. Our results demonstrated a reduction of hyperplasia of red pulp and follicular area of white pulp, increased mRNA expression of IFN-γ, TNF-α, IL-12 and iNOS, and decreased IL-10 and TGF-ß1, and intense reduction of splenic parasitism in dogs treated with the LBMPL vaccine. These results possibly suggest that the pro-inflammatory environment promoted the progressive organization of the splenic architecture favoring the cellular activation, with consequent parasite control. Along with previously obtained data, our results propose the LBMPL vaccine as a possible treatment strategy for canine visceral leishmaniasis (CVL).

Immunoprophylaxis using polypeptide chimera vaccines plus adjuvant system promote Th1 response controlling the spleen parasitism in hamster model of visceral leishmaniasis.

In recent years, several advances have been observed in vaccinology especially for neglected tropical diseases (NTDs). One of the tools employed is epitope prediction by immunoinformatic approaches that reduce the time and cost to develop a vaccine. In this scenario, immunoinformatics is being more often used to develop vaccines for NTDs, in particular visceral leishmaniasis (VL) which is proven not to have an effective vaccine yet. Based on that, in a previous study, two predicted T-cell multi-epitope chimera vaccines were experimentally validated in BALB/c mice to evaluate the immunogenicity, central and effector memory and protection against VL. Considering the results obtained in the mouse model, we assessed the immune response of these chimeras inMesocricetus auratushamster, which displays, experimentally, similar pathological status to human and dog VL disease. Our findings indicate that both chimeras lead to a dominant Th1 response profile, inducing a strong cellular response by increasing the production of IFN-γ and TNF-α cytokines associated with a decrease in IL-10. Also, the chimeras reduced the spleen parasite load and the weight a correlation between protector immunological mechanisms and consistent reduction of the parasitic load was observed. Our results demonstrate that both chimeras were immunogenic and corroborate with findings in the mouse model. Therefore, we reinforce the use of the hamster as a pre-clinical model in vaccination trials for canine and human VL and the importance of immunoinformatic to identify epitopes to design vaccines for this important neglected disease.

Development of an immunogen containing CD4+/CD8+ T-cell epitopes for the prophylaxis of tegumentary leishmaniasis.

Tegumentary leishmaniasis (TL) is a disease of high severity and incidence in Brazil, and Leishmania braziliensis is its main etiological agent. The inefficiency of control measures, such as high toxicity and costs of current treatments and the lack of effective immunoprophylactic strategies, makes the development of vaccines indispensable and imminent. In this light, the present work developed a gene encoding multiple T-cell (CD4+/CD8+) epitope, derived from conserved proteins found in Leishmania species and associated with TL, to generate a chimeric protein (rMEP/TL) and compose a vaccine formulation. For this, six T-cell epitopes were selected by immunoinformatics approaches from proteins present in the amastigote stage and associated with host-parasite interactions. The following formulations were then tested in an L. braziliensis murine infection model: rMEP/TL in saline or associated with MPLA-PHAD®. Our data revealed that, after immunization (three doses; 14-day intervals) and subsequent challenging, rMEP/TL and rMEP/TL + MPLA-vaccinated mice showed an increased production of key immunological biomarkers of protection, such as IgG2a, IgG2a/IgG1, NO, CD4+, and CD8+ T-cells with IFN-γ and TNF-α production, associated with a reduction in CD4+IL-10+ and CD8+IL-10+ T-cells. Vaccines also induced the development of central (CD44highCD62Lhigh) and effector (CD44highCD62Llow) memory of CD4+ and CD8+ T-cells. These findings, associated with the observation of lower rates of parasite burdens in the vaccinated groups, when compared to the control groups, suggest that immunization with rMEP/TL and, preferably, associated with an adjuvant, may be considered an effective tool to prevent TL. KEY POINTS: • Rational design approaches for vaccine development. • Central and effector memory of CD4+ and CD8+ T-cells. • Vaccine comprised of rMEP/TL plus MPLA as an effective tool to prevent TL.

Immunochemotherapy for visceral leishmaniasis: combinatorial action of Miltefosine plus LBSapMPL vaccine improves adaptative Th1 immune response with control of splenic parasitism in experimental hamster model.

The control of human visceral leishmaniasis (VL) is hard since there are no vaccines available as well as the treatment is hampered by toxicity and resistant parasites. Furthermore, as human, and canine VL causes immunosuppression, the combination of drugs with immunostimulatory agents is interesting to upregulate the immunity, reducing side-effects, improving treatment approaches against disease. Herein, we assessed the immunochemotherapy using miltefosine along with a vaccine formulated by Leishmania braziliensis antigens + saponin + monophosphoryl lipid-A (LBSapMPL) in L. infantum-infected hamsters. Two months after infection, the animals received treatments, and after 15 days they were evaluated for the treatment effect. The potential anti-Leishmania effect of miltefosine + LBSapMPL-vaccine was revealed by a specific immune response activation reflecting in control of spleen parasitism using half the miltefosine treatment time. The treated animals also showed an increase of total and T-CD4 splenocytes producing IFN-γ and TNF-α and a decrease of interleukin-10 and anti-Leishmania circulating IgG. In addition, it was demonstrated that the control of spleen parasitism is related to the generation of a protective Th1 immune response. Hence, due to the combinatorial action of miltefosine with LBSapMPL-vaccine in immunostimulating and controlling parasitism, this immunochemotherapy protocol can be an important alternative option against canine and human VL.

Comparative evaluation of meglumine antimoniate encapsulated in a mixture of conventional and PEGylated liposomes and immunotherapy using an anti-canine IL-10 receptor-blocking monoclonal antibody on canine visceral leishmaniasis.

This study compared the therapeutic potential of the chemotherapy using meglumine antimoniate encapsulated in a mixture of conventional and PEGylated liposomes (Nano Sbv) and immunotherapy with anti-canine IL-10 receptor-blocking monoclonal antibody (Anti IL-10R) on canine visceral leishmaniasis (CVL). Twenty mongrel dogs naturally infected by L. infantum, displaying clinical signs of visceral leishmaniasis were randomly divided in two groups. In the first one, nine dogs received six intravenous doses of a mixture of conventional and PEGylated liposomes containing meglumine antimoniate at 6.5 mg Sb/kg/dose. In the second one, eleven dogs received two intramuscular doses of 4 mg of anti-canine IL-10 receptor-blocking monoclonal antibody. The animals were evaluated before (T0) and 30, 90, and 180 days after treatments. Our major results demonstrated that both treatments were able to maintain hematological and biochemical parameters, increase circulating T lymphocytes subpopulations, increase the IFN-γ producing T-CD4 lymphocytes, restore the lymphoproliferative capacity and improve the clinical status. However, although these improvements were observed in the initial post-treatment times, they did not maintain until the end of the experimental follow-up. We believe that the use of booster doses or the association of chemotherapy and immunotherapy (immunochemotherapy) is promising to improve the effectiveness of treating CVL for improving the clinical signs and possibly reducing the parasite burden in dogs infected with Leishmania infantum.

IL-10 receptor blockade controls the in vitro infectivity of Leishmania infantum and promotes a Th1 activation in PBMC of dogs with visceral leishmaniasis.

An important strategy to reduce the risk of visceral leishmaniasis (VL) in humans is to control the infection and disease progression in dogs, the domestic reservoir of Leishmania infantum parasites. Certain therapeutic strategies that modulate the host immune response show great potential for the treatment of experimental VL, restoring the impaired effector functions or decreasing host excessive responses. It is known that the overproduction of interleukin-10 (IL-10) promotes parasite replication and disease progression in human VL as well as in canine visceral leishmaniasis (CVL). Thus, in the present study we investigated the potential of the anti-canine IL-10 receptor-blocking monoclonal antibody (Bloq IL-10R) to control and reduce in vitro infectivity of L. infantum and improve the ability of PBMC isolated from VL dogs to alter the lymphoproliferative response and intracytoplasmic cytokines. Overall, GFP+Leishmania showed lower capacity of in vitro infectivity in the presence of Bloq IL-10R. Moreover, addition of Bloq IL-10R in cultured PBMC enhanced T-CD4 and CD8 proliferative response and altered the intracytoplasmic cytokine synthesis, reducing CD4+IL-4+ cells and increasing CD8+IFN-γ+ cells after specific antigen stimulation in PBMC of dogs. Furthermore, we observed an increase of TNF-α levels in supernatant of cultured PBMC under IL-10R neutralizing conditions. Together, our findings are encouraging and reaffirm an important factor that could influence the effectiveness of immune modulation in dogs with VL and suggest that blocking IL-10R activity has the potential to be a useful approach to CVL treatment.

A chimeric vaccine combined with adjuvant system induces immunogenicity and protection against visceral leishmaniasis in BALB/c mice.

In Brazil, canine visceral leishmaniasis is an important public health problem due to its alarming growth. The high prevalence of infected dogs reinforces the need for a vaccine for use in prophylactic vaccination campaigns. In the present study, we evaluate the immunogenicity and protection of the best dose of Chimera A selected through the screening of cytokines production important in disease. BALB/c mice were vaccinated subcutaneously with three doses and challenged intravenously with 1 × 107L. infantum promastigotes. Spleen samples were collected to assess the intracellular cytokine profile production, T cell proliferation and parasite load. At first, three different doses of Chimera A (5 µg, 10 µg and 20 µg) were evaluated through the production of IFN-γ and IL-10 cytokines. Since the dose of 20 µg showed the best results, it was chosen to continue the study. Secondarily, Chimera A at dose of 20 µg was formulated with Saponin plus Monophosphoryl lipid A. Vaccination with Chimera A alone and formulated with SM adjuvant system was able to increase the percentage of the proliferation of specific T lymphocytes and stimulated a Th1 response with increased levels of IFN-γ, TNF-α and IL-2, and decreased of IL-4 and IL-10. The vaccine efficacy through real-time PCR demonstrated a reduction in the splenic parasite load in animals that received Chimera A formulated with the SM adjuvant system (92%). Additionally, we observed increased levels of nitric oxide in stimulated-culture supernatants. The Chimera A formulated with the SM adjuvant system was potentially immunogenic, being able to induce immunoprotective mechanisms and reduce parasite load. Therefore, the use of T-cell multi-epitope vaccine is promising against visceral leishmaniasis.

Establishment of monoclonal antibodies to evaluate the cellular immunity in a hamster model of L infantum infection.

Syrian hamsters (Mesocricetus auratus) are largely used as a model for infectious diseases because it is very susceptible to several pathogens, including Leishmania spp. parasites. However, the research community faces limitations in its use due to the lack of immunological reagents and tools to study the immune system in this model. In this context, we proposed the validation of some important commercially anti-mouse mAbs (CD4, TNF-α, IFN-γ and IL-10) and how this could be useful to evaluate a specific cellular immune response in Leishmania-infected hamster using flow cytometry experiments. Our data demonstrated a cross-reactivity between these anti-mouse mAbs and hamster molecules that were herein studied. Beyond that, it was able to characterize the development of a specific cellular immune response through cytokine production in L infantum-infected hamsters when compared to uninfected ones. These data not only aid the usage of hamsters as experimental model to investigate various infectious diseases, but they contribute to the design of novel approaches to further investigate the immunological mechanisms associated to pathogen infections.

Heterologous vaccine therapy associated with half course of Miltefosine promote activation of the proinflammatory response with control of splenic parasitism in a hamster model of visceral leishmaniasis.

Visceral leishmaniasis (VL) is a serious and neglected disease present worldwide. Chemotherapy using pentavalent antimony (SbV) is the most practical and inexpensive strategy available for the VL treatment today, however, it has high toxicity. Alternatively, other drugs are used as viable leishmanicidal therapeutic options. Miltefosine is the only anti-leishmanial agent administered orally, however, it has been reducing its effectiveness. In this sense, there is no ideal therapy for VL since the drugs currently used trigger severe side effects causing discontinuation of treatment, which carries an imminent risk for the emergence of parasite resistance. With that, other therapeutic strategies are gaining prominence. Among them, immunotherapy and/or immunochemotherapy, which the activation/modulation of the immune system can redirect the host's immune response to an effective therapeutic result. Therefore, this work was designed to assess an immunochemotherapy protocol composed of half course of Miltefosine associated with LBSap vaccine (Milt+LBSap) using the hamster Mesocricetus auratus as an experimental model for VL treatment. When evaluating the main hematobiochemical, immunological and therapeutic efficacy parameters, it was demonstrated that the treatment with Milt+LBSap showed restoration of hematobiochemical condition and reduced serum levels of IgG-anti-Leishmania compared to animals infected non treated (INT). Beyond that, an increase in the number of CD4+ lymphocytes producers of IFN-γ in relation to INT or to animals treated with miltefosine during 28 days, and TNF-α increased compared to INT were observed. Also, it was found a reduction of IL-10-production in relation to INT, or animals that received LBSap vaccine only, or miltefosine, following by a reduction in the splenic parasitic burden. These results demonstrate that the immunochemotherapy protocol used can stimulate the immune response, inducing an expressive cellular response sufficient to control spleen parasitism, standing out as a promising proposal for the VL treatment.

Phase I and II Clinical Trial Comparing the LBSap, Leishmune®, and Leish-Tec® Vaccines against Canine Visceral Leishmaniasis.

In this study, we performed a phase I and II clinical trial in dogs to evaluate the toxicity and immunogenicity of LBSap-vaccine prototype, in comparison to Leishmune® and Leish-Tec® vaccines. Twenty-eight dogs were classified in four groups: (i) control group received 1 mL of sterile 0.9% saline solution; (ii) LBSap group received 600 µg of Leishmania braziliensis promastigotes protein and 1 mg of saponin adjuvant; (iii) Leishmune®; and (iv) Leish-Tec®. The safety and toxicity of the vaccines were measured before and after three immunizations by clinical, biochemical, and hematological parameters. The clinical examinations revealed that some dogs of LBSap and Leishmune® groups presented changes at the site of vaccination inoculum, such as nodules, mild edema, and local pain, which were transient and disappeared seventy-two hours after vaccination, but these results indicate that adverse changes caused by the immunizations are tolerable. The immunogenicity results demonstrate an increase of B lymphocytes CD21+ regarding the Leishmune® group and monocytes CD14+ concerning LBSap and Leishmune® groups. In the in vitro analyses, an increase in lymphoproliferative activity in LBSap and Leishmune® groups was observed, with an increase of antigen-specific CD4+ and CD8+ T lymphocytes in the LBSap group. A second approach of in vitro assays aimed at evaluating the percentage of antigen-specific CD4+ and CD8+ T lymphocytes producers of IFN-γ and IL-4, where an increase in both IFN-γ producing subpopulations in the LBSap group was observed, also showed an increase in IFN-γ producers in CD8+ lymphocytes in the Leish-Tec® group. Our data regarding immunogenicity indicate that the vaccination process, especially with the LBSap vaccine, generated a protective immune response compatible with L. infantum parasite control. Based on the foregoing, the LBSap vaccine would be suitable for further studies of phase III clinical trial in endemic areas with high prevalence and incidence of canine visceral leishmaniasis (VL) cases.

Liver infusion tryptose (LIT): the best choice for growth, viability, and infectivity of Leishmania infantum parasites.

Leishmania spp. parasites have a complex biological cycle presenting basically two different morphological stages, the amastigote and promastigote forms. In vitro cultivation allows a more complete study of the biological aspects of these parasites, indicating better conditions for infection, immunoassay tests, drug evaluations, and vaccines. Thus, we evaluated the three most used culture media for Leishmania spp., Grace's insect cell culture medium (Grace's), liver infusion tryptose (LIT), and Schneider's insect medium (Schneider's), without supplementation or supplemented with fetal calf serum (FCS) and bovine serum albumin (Albumin) to evaluate the growth, viability, and infectivity of the L. infantum promastigotes. It was observed that promastigote forms have a better growth in LIT and Schneider's with or without FCS when compared to that in Grace's. The supplementation with albumin promoted greater viability of the parasites independent of the medium. For in vitro infection of J774.A1 macrophages using light microscopy and flow cytometry analyses, FCS-supplemented LIT and Grace's promoted higher percentage of infected macrophages and parasite load compared with Schneider's media. Taken together, our results demonstrated that the supplementation of LIT culture medium with FCS is the most suitable strategy to cultivate Leishmania infantum parasites enabling the maintenance of growth and infective parasites for research uses.

Histopathological changes in the gastrointestinal tract and systemic alterations triggered by experimental oral infection with Trypanosoma cruzi.

Chagas disease, caused by the protozoan Trypanosoma cruzi, is endemic in almost all countries of Latin America. In Brazil, oral infection is becoming the most important mechanism of transmission of the disease in several regions of the country. The gastrointestinal tract is the gateway for the parasite through this route of infection, however, little is known about the involvement of these organs related to oral route. In this sense, the present study evaluated the impact of oral infection on the digestive tract in mice infected by Berenice-78 (Be-78) T. cruzi strain, in comparison with the intraperitoneal route of infection. In this work, the intraperitoneal route group showed a peak of parasitemia similar to the oral route group, however the mortality rate among the orally infected animals was higher when compared to intraperitoneal route. By analyzing the frequency of blood cell populations, differences were mainly observed in CD4+ T lymphocytes, and not in CD8+, presenting an earlier reduction in the number of CD4+ T cells, which persisted for a longer period, in the animals of the oral group when compared with the intraperitoneal group. Animals infected by oral route presented a higher tissue parasitism and inflammatory infiltrate in stomach, duodenum and colon on the 28th day after infection. Therefore, these data suggest that oral infection has a different profile of parasitological and immune responses compared to intraperitoneal route, being the oral route more virulent and with greater tissue parasitism in organs of the gastrointestinal tract evaluated during the acute phase.

Recent advances and new strategies on leishmaniasis treatment.

Leishmaniasis is one of the most important tropical neglected diseases according to the World Health Organization. Even after more than a century, we still have few drugs for the disease therapy and their great toxicity and side effects put in check the treatment control program around the world. Moreover, the emergence of strains resistant to conventional drugs, co-infections such as HIV/Leishmania spp., the small therapeutic arsenal (pentavalent antimonials, amphotericin B and formulations, and miltefosine), and the low investment for the discovery/development of new drugs force researchers and world health agencies to seek new strategies to combat and control this important neglected disease. In this context, the aim of this review is to summarize new advances and new strategies used on leishmaniasis therapy addressing alternative and innovative treatment paths such as physical and local/topical therapies, combination or multi-drug uses, immunomodulation, drug repurposing, and the nanotechnology-based drug delivery systems.Key points• The treatment of leishmaniasis is a challenge for global health agencies.• Toxicity, side effects, reduced therapeutic arsenal, and drug resistance are the main problems.• New strategies and recent advances on leishmaniasis treatment are urgent.• Immunomodulators, nanotechnology, and drug repurposing are the future of leishmaniasis treatment.

Recent advances and new strategies in Leishmaniasis diagnosis.

Leishmaniasis is a set of complex and multifaceted syndromes, with different clinical manifestations, caused by different species of the genus Leishmania spp. that can be characterized by at least four syndromes: visceral leishmaniasis (VL, also known as kala-azar), post-kala-azar dermal leishmaniasis (PKDL), cutaneous leishmaniasis (CL), and mucocutaneous leishmaniasis (MCL). Among the most serious clinical forms, VL stands out, which causes the death of around 59,000 people annually. Fast and accurate diagnosis in VL is essential to reduce the disease's morbidity and mortality. There are a large number of diagnostic tests for leishmaniasis, however they do cross-react with other protozoa and their sensitivity changes according to the clinical form of the disease. Thus, it is essential and necessary to provide a diagnosis that is sufficiently sensitive to detect asymptomatic infected individuals and specific to discriminate individuals with other infectious and parasitic diseases, thus enabling more accurate diagnostic tools than those currently used. In this context, the aim of this review is to summarize the conventional diagnostic tools and point out the new advances and strategies on visceral and cutaneous leishmaniasis diagnosis.

Chimeric Vaccines Designed by Immunoinformatics-Activated Polyfunctional and Memory T Cells That Trigger Protection against Experimental Visceral Leishmaniasis.

Many vaccine candidates against visceral leishmaniasis (VL) have been proposed; however, to date, none of them have been efficacious for the human or canine disease. On this basis, the design of leishmaniasis vaccines has been constantly changing, and the use of approaches to select specific epitopes seems to be crucial in this scenario. The ability to predict T cell-specific epitopes makes immunoinformatics an even more necessary approach, as in VL an efficient immune response against the parasite is triggered by T lymphocytes in response to Leishmania spp. immunogenic antigens. Moreover, the success of vaccines depends on the capacity to generate long-lasting memory and polyfunctional cells that are able to eliminate the parasite. In this sense, our study used a combination of different approaches to develop potential chimera candidate vaccines against VL. The first point was to identify the most immunogenic epitopes of Leishmania infantum proteins and construct chimeras composed of Major histocompatibility complex (MHC) class I and II epitopes. For this, we used immunoinformatics features. Following this, we validated these chimeras in a murine model in a thorough memory study and multifunctionality of T cells that contribute to a better elucidation of the immunological protective mechanisms of polyepitope vaccines (chimera A and B) using multicolor flow cytometry. Our results showed that in silico-designed chimeras can elicit polyfunctional T cells producing T helper (Th)1 cytokines, a strong immune response against Leishmania antigen, and the generation of central and effector memory T cells in the spleen cells of vaccinated animals that was able to reduce the parasite burden in this organ. These findings contribute two potential candidate vaccines against VL that can be used in further studies, and help in this complex field of vaccine development against this challenging parasite.

Synthetic Peptides Elicit Strong Cellular Immunity in Visceral Leishmaniasis Natural Reservoir and Contribute to Long-Lasting Polyfunctional T-Cells in BALB/c Mice.

Reverse vaccinology or immunoinformatics is a computational methodology which integrates data from in silico epitope prediction, associated to other important information as, for example, the predicted subcellular location of the proteins used in the design of the context of vaccine development. This approach has the potential to search for new targets for vaccine development in the predicted proteome of pathogenic organisms. To date, there is no effective vaccine employed in vaccination campaigns against visceral leishmaniasis (VL). For the first time, herein, an in silico, in vitro, and in vivo peptide screening was performed, and immunogenic peptides were selected to constitute VL peptide-based vaccines. Firstly, the screening of in silico potential peptides using dogs naturally infected by L. infantum was conducted and the peptides with the best performance were selected. The mentioned peptides were used to compose Cockt-1 (cocktail 1) and Cockt-2 (cocktail 2) in combination with saponin as the adjuvant. Therefore, tests for immunogenicity, polyfunctional T-cells, and the ability to induce central and effector memory in T-lymphocytes capacity in reducing the parasite load on the spleen for Cockt-1 and Cockt-2 were performed. Among the vaccines under study, Cockt-1 showed the best results, eliciting CD4+ and CD8+ polyfunctional T-cells, with a reduction in spleen parasitism that correlates to the generation of T CD4+ central memory and T CD8+ effector memory cells. In this way, our findings corroborate the use of immunoinformatics as a tool for the development of future vaccines against VL.